Shear stress is required for the endocytic uptake of the factor VIII‐von Willebrand factor complex by macrophages

Abstract

Low-density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)-von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte-derived macrophages. A confocal microscopy study was employed to study the uptake by monocyte-derived macrophages of a functional green fluorescent FVIII-GFP derivative in the presence and absence of VWF. The results revealed that FVIII-GFP is internalized by macrophages. We found that FVIII-GFP co-localizes with LDL receptor-related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII-GFP. However, FVIII-GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII-VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII-GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII-GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII-GFP and VWF was inhibited. The results show that shear stress is required for macrophages to internalize both constituents of the FVIII-VWF complex.